Our long-term goal is to understand the structural basis of transcriptional regulation -- how regulatory proteins recognize their DNA-binding sites and how they interact with each other when bound to DNA. We will pursue these aims by refining our understanding of the bacteriophage 434 repressor and Cro proteins and by extending our X-ray crystallographic analysis to the GAL4 family of yeast transcriptional activators. (1) The 434 genetic switch. In order to complete our detailed picture of this switch, we will determine, at high resolution (at least 2.5 degrees Alpha) a series of structures from crystals of complexes between R1-69 (the DNA-binding domain of 434 repressor) and operator-containing DNA fragments as well as complexes between 434 Cro and similar DNA fragments. We will also attempt to design a repressor with completely Cro-like binding ratios and to design operators with predictable affinities. These molecules will be produced and their properties tested. We will attempt to visualize cooperative interactions by crystallizing intact repressor on an OR1-plus-OR2 containing DNA segment. (2) GAL4. We will attempt to crystallize the N-terminal part (residues 1-147) of GAL4, in complex with DNA containing the 17 base-pair UASG binding site. We will measure binding of GAL4 or GAL4(1-147) to variant UASG sequences, systematically altered at selected positions. The 80-residue, highly acidic activating domain from Herpes simplex virus VP16 functions when attached to Gal4(1-147). We will determine whether this domain is well folded and hence whether it can be crystallized, either independently or attached to GAL4; a very long range goal is to study such a domain in complex with its functional target.